Systems and methods for processing fluids

ABSTRACT

Systems and methods for processing fluid samples are disclosed. Fluid sample processing is accomplished using a series of microfluidic bump arrays include an automated and integrated system for sorting particles from a biological sample, lysing those particles to expose total RNA or DNA, purifying the RNA or DNA, processing the RNA or DNA by chemical or enzymatic modification, to select RNA or DNA molecules by size, or to generate, optionally, a sequencing library. The sequencing library is suitable for use in next generation sequencing (“NGS”).

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional PatentApplication No. 61/515,063 to Boles, filed Aug. 4, 2011, and entitled“Microfluidic Bump Array,” and incorporates its disclosure herein byreference in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to the field of molecularbiology, and in particular to systems and methods that can be used toisolate high molecular weight Ribonucleic Acid (“RNA”) andDeoxyribonucleic Acid (“DNA”) from biological samples, wherein the RNAand DNA molecules can be subsequently sorted by size and/or used togenerate sequencing libraries.

BACKGROUND

Next-generation sequencing (“NGS”) has revolutionized research in manyareas of molecular biology, genetics, and medicine. As NGS technologyhas become more affordable and more widely available over the past fewyears, there has been increasing focus on the need for more efficientand reproducible sample preparation methods for NGS library generation.Conventional methods involve many cycles of enzymatic modificationfollowed by purification, an arrangement that is laborious,time-consuming, and prone to template loss.

Conventional processing and purification methods in molecular biologyinvolve nucleic acids undergoing sequential cycles of treatment followedby purification, wherein treatment and purifications are usually carriedout in a separate tubes or vessels, and the overall workflow involvesrepeated liquid transfers (by manual or robotic pipetting devices)between the different reaction vessels. In conventional workflows, eachpurification step typically involves removal of the nucleic acids fromthe previous reaction mixture by chemical extraction, precipitation,and/or adsorption to solid phases (such as microparticles or filters).Because of the inefficiencies in the multiple liquid transfer andpurification steps, poor sample yield and loss of samples due to usererror are major problems for complex molecular biology workflows (likethose used in NGS).

SUMMARY

In some implementations, the current subject matter provides a systemand method for microfluidic sample preparation. The preparation can beaccomplished through the use of a single continuous flow technology,referred to as a “bump array”, also referred to as determininsticlateral displacement (“DLD”), that can be used to manipulate andseparate cells, organelles, microparticles, and high molecular weight(“HMW”) deoxyribonucleic acid (“DNA”) molecules that exhibitparticle-like properties.

In some implementations, the current subject matter relates to a methodfor processing of a biological fluid. The method can include separatingat least one first cell from the biological fluid, applying at least onefirst treatment to the at least one separated cell to produce a firsttreated solution, applying at least one second treatment to the firsttreated solution to produce a second treated solution, and processing atleast one of the first treated solution and the second treated solutionusing a deterministic lateral displacement to generate an outputsolution.

In some implementations, the current subject matter can include one ormore of the following optional features. The biological fluid caninclude at least one of the following: whole blood, urine, spinal fluid,saliva, buccal swabs, sputum, bronchial lavage, gastric lavage fluid,microbial culture media, feces, buffy coat, serum, plasma, plateletconcentrate, water samples, and/or any other biological, chemical,and/or biochemical fluids and/or any combination thereof. Thedeterministic lateral displacement can use at least one bump array toprocess at least one of the first treated solution and the secondtreated solution. The deterministic lateral displacement can use asequential arrangement of a plurality of bump arrays to process at leastone of the first treated solution and the second treated solution.

In some implementations, the biological fluid can be whole blood. Theapplying of at least one first treatment can include lysing cellsseparated from the whole blood to generate a purified deoxyribonucleicacid (“DNA”). The applying of at least one second treatment can includecombining the purified DNA with a transposase complex and at least onesequencing adaptor.

In some implementations, the method can further include fractionatingthe output solutions based on a size of at least one cell containedwithin the output solution.

In some implementations, the current subject matter can relate to asystem for processing of a biological fluid. The system can include atleast one input reservoir for receiving the biological fluid andseparating at least one first cell from the biological fluid, at leastone bump array mechanism coupled to the at least one input reservoir forapplying at least one first treatment to the at least one separated cellto produce a first treated solution, applying at least one secondtreatment to the first treated solution to produce a second treatedsolution, and processing at least one of the first treated solution andthe second treated solution using a deterministic lateral displacementto generate an output solution, and an output reservoir for receivingthe output solution. In some implementations, the current subject mattercan include various optional features discussed above and in thefollowing text of the present disclosure.

In some implementations, the current subject matter relates to a methodfor processing a whole blood sample using a sequential and continuousarrangement of bump arrays integrated in a continuous flow operation.The method can include receiving the whole blood sample at a first bumparray in the arrangement of bump arrays, purifying the whole bloodsample to produce white blood cells, isolating nuclei from the whiteblood cells, isolating deoxyribonucleic acid (“DNA”) from the nuclei,purifying DNA from the nuclei, and treating the purified DNA using atleast one chemical and/or enzymatic DNA treatment.

In some implementations, the current subject matter relates to a methodfor processing of a fluid sample using at least one bump array. Themethod can include receiving the fluid sample at the at least one bumparray, isolating, using the at least one bump array, at least onenucleic acid-containing cell and/or particle of interest from the fluidsample on the basis of a size of the cell and/or particle, contacting,using the at least one bump array, the isolated cell and/or particlewith at least one reagent stream for releasing at least one nucleic acidfrom the cell and/or particles in substantially pure form, moving, usingthe at least one bump array, the at least one purified nucleic acids outof the reagent stream, and removing the at least one purified nucleicacid from the at least one bump array.

In some implementations, the current subject matter can include one ormore of the following optional features. A plurality of bump arrays canprocess the nucleic acid, wherein individual bump arrays in theplurality of bump arrays can be connected in series, wherein an outputof one bump array can be provided to an input of a subsequent individualbump array. Alternatively, a single bump array can be used for thereceiving, the isolating, the contacting and the moving. The fluidsample can include at least one of the following: an avian whole bloodand a mammalian whole blood and the nucleic acid-containing cells and/orparticles can be white blood cells. The fluid sample can include atleast one of the following: an avian whole blood and a mammalian wholeblood, and the nucleic acid-containing cells and/or particles can becirculating tumor cells. The fluid sample can include at least one ofthe following: an avian whole blood and a mammalian whole blood, and thenucleic acid-containing cells and/or particles can include at least oneof the following: white blood cells, bacteria, viruses, fungi, andparasitic protozoans.

The biological fluid can include at least one of the following: wholeblood, urine, spinal fluid, saliva, buccal swabs, sputum, bronchiallavage, gastric lavage fluid, microbial culture media, feces, buffycoat, serum, plasma, platelet concentrate, water samples, and/or anyother biological, chemical, and/or biochemical fluids and/or anycombination thereof.

In some implementations, the current subject matter relates to a methodfor serially processing of a high molecular weight (“HMW”) nucleic acidusing at least one chemical and/or enzymatic reagent stream using atleast one bump array, wherein HMW nucleic acid has an effectivehydrodynamic radius that is greater than a critical size of the at leastone bump array. The method can include receiving the HMW nucleic acid atthe at least one bump array, and contacting the HMW nucleic acid withthe at least one chemical and/or enzymatic reagent stream, wherein theat least one chemical and/or enzymatic reagent stream flows in thedirection of bulk fluid flow through the bump array, whereas the HMWnucleic acid flows at an angle to the direction of bulk fluid flow. TheHMW nucleic acid can react with the at least one chemical and/orenzymatic reagent stream.

In some implementations, the current subject matter relates to a methodfor serial processing of a nucleic acid using at least one chemicaland/or enzymatic reagent stream using at least one bump array. Themethod can include receiving the nucleic acid, flowing the nucleic acidinto the at least one bump array, contacting, using the at least onebump array, the nucleic acid with at least one chemical and/or enzymaticreagent stream, modifying, using at least one chemical and/or enzymaticreagent stream, the nucleic acid, and removing, using the at least onebump array, the purified nucleic acid from the at least one chemicaland/or enzymatic reagent stream.

In some implementations, the current subject matter can include one ormore of the following optional features. A plurality of bump arrays canserially process the nucleic acid, wherein individual bump arrays in theplurality of bump arrays can be connected in series, wherein an outputof one bump array in the plurality of bump arrays can be input to thesubsequent individual bump array in the plurality of bump arrays.Alternatively, a single bump array can perform the flowing, thecontacting, the modifying, and the removing. The nucleic acid can be ahigh molecular weight (“HMW”) nucleic acid. Alternatively, the nucleicacid can be a deoxyribonucleic acid (“DNA”), wherein the DNA can bebound to at least one microparticle for carrying the DNA through thebump array. The DNA can be bound using at least one of the following:covalent binding and non-covalent binding.

In some implementations, the current subject matter relates to a methodfor processing of a fluid sample using at least one bump array. Themethod can include receiving the fluid sample at the at least one bumparray, isolating, using the at least one bump array, at least onenucleic acid-containing cell and/or particle of interest from the fluidsample on the basis of a size of the cell and/or particle, contacting,using the at least one bump array, the isolated cell and/or particlewith at least one reagent stream for releasing at least one nucleic acidfrom the cell and/or particles in substantially pure form, modifying thenucleic acid, and moving, using the at least one bump array, the atleast one purified nucleic acid out of the reagent stream, and removingthe at least one purified nucleic acid from the at least one bump array.

In some implementations, the current subject matter can include one ormore of the following optional features. The nucleic acid can be a highmolecular weight (“HMW”) nucleic acid. Alternatively, the nucleic acidcan be a deoxyribonucleic acid (“DNA”), wherein the DNA can be bound toat least one microparticle for carrying the DNA through the bump array.The DNA can be bound using at least one of the following: covalentbinding and non-covalent binding. The fluid sample can be seriallyprocessed using at least one chemical and/or enzymatic reagent streamusing the at least one bump array. The nucleic acid can be modifiedusing at least one chemical and/or enzymatic reagent stream. Themodified nucleic acid can include at least one of the following: adeoxyribonucleic acid (“DNA”) sequencing library and a recombinant DNAlibrary.

In some implementations, the current subject matter relates to a reagentsystem for generating DNA sequencing libraries. The system can include atransposase reagent complexed with a linear DNA reagent, the DNA reagenthaving transposase recognition sequences and sequencing adaptersequences at each end of the DNA reagent, whereby on reaction with a DNAmolecule targeted for sequencing, the transposase inserts theadapter-bearing linear DNA reagent into the sequencing target to form acointegrate structure, wherein the sequencing target is cleaved at asingle position, and the ends of the cleaved target are joined to theends of the adapter-bearing linear DNA reagent.

In some implementations, the use of a singular separation technologythat can accommodate multiple types of particles (e.g., cells,organelles, microparticles, and HMW DNA molecules) can provide forexample, but not limited to, an ability to accomplish multiplesequential processing steps by a common process, in a single operation,and on a single consumable device. Further, in multi-step processes,integration of reaction and post-reaction cleanup steps can enableseamless, substantially zero-loss transfer of sample between processingsteps. Sample purification can be accomplished on the basis of“particle” size alone. Thus, differential adsorption to a solid phase isnot used and sample loss due to irreversible adsorption can be avoided.In some implementations, a portion of the initial sample that isretained in the system and either passed onto a further processing orcollected at the end, can be purified after every step in each processby mechanisms, including, but not limited to, buffer exchange andremoval of low molecular weight (“LMW”) reagents. The current subjectmatter system can provide a hands-free means for a lengthy and complexsample preparation process that may be required for NGS. All of theprocesses can be performed by current subject matter systems and methodsubstantially without user intervention, and, thus, user-mediated errorand user-mediated variability can be substantially obviated. Bump arrayNGS processing can be used for routine, quality-control (“QC”)-intensiveapplications like clinical trials and diagnostic testing.

In principle, input sample size can be scaled from 100's of microliters(“μl”) of whole blood (e.g., 100 μl to 999 μl of whole blood) down tothe single cell level, a feature that may accelerate sequencingapplications in cancer research and diagnostics.

The details of one or more variations of the subject matter describedherein are set forth in the accompanying drawings and the descriptionbelow. Other features and advantages of the subject matter describedherein will be apparent from the description and drawings, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, show certain aspects of the subject matterdisclosed herein and, together with the description, help explain someof the principles associated with the disclosed implementations. In thedrawings,

FIG. 1 is a schematic diagram illustrating an exemplary bump array;

FIG. 2A is a photograph illustrating fluorescent microparticles 0.4 μm(green) and 1.0 μm (red) flowing through a bump array, λ=8 μm, λ/d=10;

FIG. 2B is a schematic diagram illustrating the trajectory of a particlehaving a smaller than the critical size for any given bump array;

FIG. 2C is a schematic diagram illustrating the trajectory of a particlehaving a greater than the critical size for any given bump array;

FIG. 3A is a schematic diagram illustrating an exemplary bump array formoving particles in and out of reagent streams, including a blow up ofthe texture of the obstacles on the chip;

FIG. 3B is a photograph of the exemplary bump array shown in FIG. 3A;

FIG. 4A-H is a series of photographs illustrating the lysis of an E.coli cell in a bump array;

FIG. 5 illustrates an exemplary system for processing of a fluid sample,according to some implementations of the current subject matter;

FIG. 6 illustrates another exemplary system for processing of a fluidsample, according to some implementations of the current subject matter;

FIG. 7 illustrates another exemplary system for processing of a fluidsample, according to some implementations of the current subject matter;

FIG. 8A is a schematic diagram illustrating a transposition-mediatedlibrary generation system optimized for use in automated bump arrayinstrument;

FIG. 8B is a schematic diagram illustrating a structure of transpositioncointegrates;

FIG. 9 illustrates in schematic form, an exemplary strategy forprocessing of a fluid sample, according to some implementations of thecurrent subject matter;

FIG. 10 is a method, according to some implementations of the currentsubject matter.

DETAILED DESCRIPTION

The discussion in the present disclosure may refer to and/or use variousterms in connection with describing various implementations of thecurrent subject matter's systems and methods. The following definitionsof such terms are provided for illustrative purposes only and are notintended to limit the scope of the current subject matter disclosedherein.

The term “sample” or “biological sample” can describe a plurality ofparticles that can be separated and processed by the bump array.Exemplary particles can include, but are not limited to, cells, nuclei,organelles, high molecular weight ribonucleic acid (“RNA”) ordeoxyribonucleic acid (“DNA”) (RNA and DNA can be collectively referredherein as nucleic acid (“NA”)), and microorganisms (e.g., bacterium andviruses) within a biological fluid or tissue. When particles areprocessed from intact tissue, such as a biopsy of a tumor or neoplasm,the cells are typically dissociated and resuspended in a fluid prior tointroducing the particles into an array or system of some embodiments ofthe disclosure.

The term “fraction” can describe a subset of the particles within asample. A fraction can be defined or determined by size. Alternatively,a fraction can be defined or determined by any physical property, suchas size, that causes it to differentially traverse the field of posts ina bump array. For instance, fractions containing particles of smallersizes can travel in pathways that more closely approximate the vectordirection in which the primary fluid or stream flows across the array(for example, as shown in FIGS. 2B, 9 and 10). In contrast, fractionscontaining particles of larger sizes can travel in pathways that deviatefurther from the vector direction in which the primary fluid or streamflows across the array (i.e., they are diverted or bumped away from themain flow direction at a more severe angle) (for example, as shown inFIGS. 2C, 9 and 10).

An exemplary “sample” can include, but is not limited to, a cell, anucleus, an organelle, a HMW RNA (intranuclear, intracellular, orextracellular), a HMW DNA (intranuclear, intracellular, orextracellular), a microorganism, a bacterium, a virus, or anycombination thereof “Biological fluids” can include, but are not limitedto, aqueous humour and vitreous humour, bile, blood (whole blood, serum,plasma, cell-rich fractions), breast milk, cerebrospinal fluid (“CSF”),endolymph and perilymph, gastric juice, mucus (including phlegm),peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat,tears, vaginal secretion, vomit, and urine. “Biological tissues” caninclude, but are not limited to, those tissues derived from theendoderm, mesoderm, or ectoderm; those tissues that can be connective,muscle, nervous, or epithelial in nature; tissues that can include bone,cartilage, tendon, bone marrow, blood, vasculature (arteries and veins),smooth muscle, skeletal muscle, cardiac muscle (the heart), the centralnervous system (brain, spinal cord, cranial nerves), peripheral nervoussystem (peripheral nerves), skin, respiratory tract, digestive tract,and reproductive tract.

Nucleic acids can be derived from genomic DNA, double-stranded DNA(“dsDNA”), single-stranded DNA (“ssDNA”), coding DNA (“cDNA”), messengerRNA (“mRNA”), short interfering RNA (“siRNA”), short-hairpin RNA(“shRNA”), microRNA (“miRNA”), single-stranded RNA, double-stranded RNA(“dsRNA”), a morpholino, RNA interference (“RNAi”) molecule,mitochondrial nucleic acid, chloroplast nucleic acid, viral DNA, viralRNA, and other organelles with separate genetic material. Furthermore,samples can include nucleic acid analogs that can contain modified,synthetic, or non-naturally occurring nucleotides or structural elementsor other alternative/modified nucleic acid chemistries known in the art.Additional examples of nucleic acid modifications can include the use ofbase analogs such as inosine, intercalators and minor groove binders.Other examples of nucleic acid analogs and alternative/modified nucleicacid chemistries can be used as well.

PNA oligomers can be included in exemplary samples or fractions of someembodiments of the disclosure. PNA oligomers can be analogs of DNA inwhich the phosphate backbone is replaced with a peptide-like backbone.

Polypeptides or proteins can be complex, three-dimensional structurescontaining one or more long, folded polypeptide chains. Polypeptidechains are composed of a plurality of small chemical units called aminoacids. Naturally occurring amino acids can have an L-configuration.Synthetic peptides can be prepared employing conventional syntheticmethods, using L-amino acids, D-amino acids or various combinations ofL- and D-amino acids. The term “peptide” can describe a combination twoor more amino acids. Naturally occurring amino acids have anL-configuration. Peptides having fewer than ten amino acids can be“oligopeptides,” whereas peptides containing a greater number of aminoacid units are “polypeptides.” Any reference to a “polypeptide” alsoincludes an oligopeptide. Further, any reference to a “peptide” caninclude polypeptides and oligopeptides. Each different arrangement ofamino acids can form a different polypeptide chain.

The term “nucleic acid molecule” can describe the phosphate esterpolymeric form of ribonucleosides (adenosine, guanosine, uridine orcytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), orany phosphoester analogues thereof, such as phosphorothioates andthioesters, in either single stranded form, or a double-stranded helix.The term “nucleic acid molecule,” and in particular DNA or RNA molecule,can refer only to the primary and secondary structure of the molecule,and does not limit it to any particular tertiary forms. Thus, this termcan include double-stranded DNA found, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. A“recombinant DNA molecule” is a DNA molecule that has undergone amolecular biological manipulation.

Nucleic acids can be processed by chemical or enzymatic reactions withinthe bump array or system of some embodiments of the disclosure to impartfluorescent, magnetic, or radioactive properties to these molecules forthe purpose of supporting sequence detection or analysis in subsequentanalyses or for use in devices other than the bump arrays and systemsdescribed herein.

Regarding polypeptides, the term “native” can describe a non-denaturedpolypeptide. Polypeptides of according to some embodiments of thedisclosure are native or denatured.

In some implementations, the current subject matter relates to systemsand methods for processing biological fluids by using a “bump array”and/or multiple “bump arrays” and for creating next-generationsequencing libraries based on the processed biological fluids. In someimplementations, the biological fluids can include, but are not limitedto, whole blood, urine, spinal fluid, saliva, buccal swabs, sputum,bronchial lavage, gastric lavage fluid, microbial culture media, feces,buffy coat, serum, plasma, platelet concentrate, water samples, and/orany other biological, chemical, and/or biochemical fluids and/or anycombination thereof. The bump array can be also referred to as adeterministic lateral displacement (“DLD”) mechanism that can separatecertain size molecules from a fluid.

In some implementations, the current subject matter can implement aseries of bump arrays that can be integrated into one continuous flowoperation, where one can use a crude biological sample as input and thenprocess the sample by purifying the sample, isolating various componentscontained in the sample, such as, for example, molecules, cells, etc.from the sample, purifying isolated components and performing varioustreatments on the purified isolated components. For exemplary,illustrative and non-limiting purposes only, the following discussionwill refer to whole blood as a biological sample being processed.However, it should be understood that the current subject matter is notlimited to the use of whole blood and can include any of the abovebiological fluids as well as any others.

Assuming that the biological sample is whole blood, then the currentsubject matter's system can perform the following operations: purifywhite blood cells (“WBC”) from the whole blood, isolate nuclei from thecells, isolate DNA from the nuclei, perform DNA purification from thenuclei, and perform various chemical and/or enzymatic DNA treatments onthe purified DNA. In some implementations, any purified nucleic acidscan be used for performance of various chemical and/or enzymatic DNAtreatments on them. In some implementations, the current subject mattercan allow a biological sample to be contacted with various reagents andremoved from the reagent stream (as needed) using DLD. Further, in someimplementations, the current subject matter can process smaller nucleicacids using bump array(s) by attaching the molecules to microparticlesthat are bumpable. In this way, the particles can be used to drag theirDNA cargo through multiple reagent streams.

In some implementations, the current subject matter system can includeat least one bump array device that can have one or more bump arrays.The bump array device can serially treat and purify nucleic acid fluidsamples. Multiple cycles of treatment and purification can be carriedout using a single flow device in a single continuous flow operation.The treatments can be chemical and/or enzymatic. The nucleic acids canbe purified from cells and/or complex liquid biological sample, such aswhole blood. The bump array device can also be used for performingvarious processing of the purified nucleic acids. Non-limiting examplesof such processing can include at least one of the following:phosphorylation, dephosphorylation, restriction digestion, ligation,denaturation, hybridization, processing by polymerases, fluorescent orradioactive labeling, chemical modification of DNA bases or backbonegroups, enzymatic or chemical excision of modified bases, staining ofnucleic acids with chromophores or fluorophores, etc. and/or othersand/or any combination thereof. The nucleic acids can be particle boundnucleic acids, where nucleic acids can be attached to microparticles.This can allow for processing of small nucleic acids. The particles canrender the attached nucleic acids bumpable in arrays with easilymanufactured array dimensions.

In some implementations, the current subject matter can provide a systemand a method for processing of fluids. The processing can includepurification of fluids which can be accomplished by flowing a complexfluid sample into a bump array, using a bump array to isolate nucleicacid-containing cells or particles of interest on the basis of particlesize, using a bump array to contact isolated particles with one or onereagent streams that can release nucleic acid from the particles insubstantially pure form, and using a bump array to move purified nucleicacids out of the reagent stream. Once the purified nucleic acids aremoved out of the reagent stream, the purified nucleic acids can besubstantially free from other cellular and sample components and can besubstantially free from reagent stream components of the bump array.

In some implementations, the individual bump arrays can be connected inseries so that the product output of one bump array can be connected tothe sample input of the subsequent individual bump arrays. In someimplementations, the same bump array can be used for all steps.Furthermore, cell fractionation and reagent treatments can beaccomplished in physically distinct regions of a single bump array. Insome implementations, the input sample can be avian or mammalian wholeblood and the nucleic-acid-containing particles can be white bloodcells. In some implementations, the input sample can be avian ormammalian whole blood and the nucleic-acid-containing particles can becirculating tumor cells. In some implementations, the input sample canbe avian or mammalian whole blood and the nucleic-acid-containingparticles can be white blood cells, bacteria, viruses, fungi, parasiticprotozoans and/or any others and/or any combination thereof.

In some implementations, the current subject matter can provide a serialprocessing of high molecular weight nucleic acids by chemical andenzymatic means on bump arrays. The HMW nucleic acid can have aneffective hydrodynamic diameter that can be greater than the criticaldiameter of the bump array and the HMW nucleic acid can be contactedwith at least a first reagent stream, where the first reagent stream canflow in the direction of bulk fluid flow and where the HMW nucleic acidis bumped through the first reagent stream and can react with the firstreagents.

In some implementations, the current subject matter can provide a serialprocessing of HMW nucleic acids by one or more chemical or enzymaticmeans that can be accomplished by flowing a sample of HMW nucleic acidsinto a bump array, using a bump array to contact HMW nucleic acids withat least one reagent streams that can modify the nucleic acids (e.g.,chemically, enzymatically, etc.), and, optionally, using a bump array toremove purified nucleic acids from the reagent stream.

In some implementations, the bump arrays can be individual bump arraysconnected in series so that the product output of one bump array can beconnected to the sample input of the subsequent individual bump arrays.The bump arrays can be the same bump arrays (and the cell fractionationand reagent treatments can be accomplished in physically distinctregions of one continuous bump array). Further, assuming that the DNAsample can be bound (covalently or noncovalently) to microparticles, themicroparticles can be bumpable and can therefore act as carriers to takethe DNA through the modification reactions.

In some implementations, the current subject matter can provide forprocessing of whole blood to produce a pure nucleic acid, which can beused to produce a modified pure nucleic acid. The modified pure nucleicacid can be a DNA sequencing library and/or a recombinant DNA library.

In some implementations, the current subject matter can provide a systemthat can accept a whole blood sample as input and produce a genomic DNAlibrary suitable for next-generation sequencing (“NGS”). Libraryconstruction can take place in a single automated process without anyuser intervention. The system can lower the cost and labor of NGSsequencing and accelerate movement of NGS technology into diagnosticsettings. The system can be scalable to accommodate samples containingvery few cells (e.g., a single cell level), which can be important intreatment of cancer and/or other important medical problems.

In some implementations, the system can include a microfluidic,continuous-flow design. Liquid samples containing particles (e.g.,cells, nuclei, and large macromolecules such as randomly-coiled HMW DNA)can be pumped through flow cells that can be populated by a regulararray of micron-sized posts. The spacing and alignment of the posts canbe arranged so that particles above a certain critical size can be“bumped” by the posts into a flow path that runs diagonally across thedirection of bulk liquid flow. In contrast, sample components smallerthan the critical size can travel straight along with the bulk flow.Using this mechanism, larger sample components can be separated andpurified from smaller components laterally across the chip.

Samples can flow through these bump arrays under conditions of laminarflow (Reynolds number, R_(e), <<1), so that discrete reagent streams canbe introduced into arrays without significant lateral mixing. Largeparticles can be bumped diagonally into, and out of, such reagentstreams to perform chemical or enzymatic reactions on the particles. Thecurrent subject matter system can use this principle to purify leukocytenuclei, purify DNA, and enzymatically modify DNA for generation of NGSlibraries.

Although the following steps are illustrated using a blood sample, thisprocess can be performed on any biological fluid and/or any tissuesample from which the corresponding cells have been dissociated from oneanother and re-suspended in a fluid (as illustrated in Table 1 below).The isolation of foreign cells from a host can also be performed. Forexample, the raw material can be whole blood and/or a cell solutionderived from whole tissue, but the intermediate fraction of interest canbe a virus, a prokaryotic cell that is not native to the host (such as abacterium), a parasite, a fungus, a pathogenic microbe, and/or any otherfraction, component, fluid, etc., and/or any combination thereof.

TABLE 1 Raw Materials, intermediates, and processed output (alsopreferred embodiments) of bump array. Raw Input (biological Preferredfluid or Cell solution Processed derived from tissue) Intermediate(s)Processed Output Output Whole Blood White Blood Cell(s) Isolated and/orpurified total NGS Library (WBC(s)) DNA Whole Blood Cell-free fractionIsolated and/or purified cell- NGS Library free DNA Tumor BiopsyIsolated and/or Isolated and/or purified total NGS Library for apurified tumor DNA from a single tumor cell single cell cell(s) BuccalSwab/spit Isolated and/or purified total NGS Library DNA High MolecularWeight Fragmented DNA Size-fractionated DNA NGS Library, (HMW) DNARecombinant Library Bacterial/eukaryotic Isolated and/or purified totalNGS Library, cultured cells DNA Recombinant Library Infected bloodMicrobial cells HMW microbial DNA NGS Library Liquid Microbial CultureMicrobial cells HMW microbial DNA NGS Library Urine Microbial cells HMWmicrobial DNA NGS Library Spinal Fluid Microbial cells HMW microbial DNANGS Library

FIG. 1 is a schematic diagram illustrating an exemplary bump array 100(an exemplary bump array is illustrated in Davis J A, Inglis D W, MortonK J, Lawrence D A, Huang L R, Chou S Y, Sturm J C, Austin R H.“Deterministic hydrodynamics: taking blood apart.” Proc Natl Acad SciUSA. vol. 103, 14779-84. 2006, which is incorporated herein by referencein its entirety). The bump array 100 can include a plurality of posts106 and a plurality of streamlines 108, where the posts can be disposedin the streamlines 108 in a predetermined fashion, in a random fashion,and/or in any other desired way. The posts 106 can be disposed apredetermined distance G from one another, thereby creating a gap allowmolecules, particles, etc. 102, 104 to move between the posts in thestreamlines 108. The posts 106 can be disposed in accordance with apredetermined horizontal spacing λ and can have a predetermined rowoffset d, as shown in FIG. 1. In some implementations of bump arrayapplications, the parameters G, λ, and/or d can be particularly selectedfor the specific dimensions of the particles being separated by the bumparray 100. Such exemplary design considerations are discussed by Ingliset al. (Inglis D W, Davis J A, Austin R H, Sturm J C. “Critical particlesize for fractionation by deterministic lateral displacement.” Lab Chip.vol. 6, 655-8. 2006, incorporated herein by reference in its entirety).As shown in FIG. 1, various size particles (e.g., small particles 102and large particles 104) can move through the bump array 100 in apredetermined fashion (as shown by the arrows).

Liquid sample with particles flows vertically through regular post array(shown by the arrow in FIG. 1), with horizontal post spacing of λ, androw offset, d, will generate λ/d liquid streamlines in each gap.Particles 104 with radius greater than the width of the first streamlineare bumped diagonally (passing from streamline 1 to 2) at every gap.Small particles 102 with a radius smaller than the width of streamline,stay in the same streamline and pass vertically down the array with nolateral displacement.

FIG. 2A is illustrating fluorescent microparticles 202 having a diameterof 0.4 μm (green) and microparticles 204 having a diameter of 1.0 μm(red) flowing through a bump array, λ=8 μm, λ/d=10. The green particles202 are smaller than the critical dimension for bumping and travelstraight down along the lines of bulk liquid flow. The particles 202 canfollow a zigzag streamline 108 path between the posts 106 with nolateral displacement, as shown in FIG. 2B. The trajectory of theparticle 202 having a smaller diameter than the critical size for thebump array 100. While the path of the particle 202 follows a zigzagpattern around the posts 106, its flow continues in same direction asthe general flow of the fluid sample being processed in the bump array100. The red particles 204 can be larger than the critical dimension andcan be bumped along a diagonal streamline 108 path across the array 100,as shown in FIG. 2C. The trajectory of the particle 204 can follow anangled trajectory with respect to the general flow of the fluid samplethrough the bump array 100, because the particles 204 can be bumpeddiagonally away from the posts 106. In some implementations, the bumparray 100 can be used for cell sorting, size selection of large DNA (>20kb), and microparticle size selection.

FIG. 3A is a schematic diagram illustrating a bump array 300 for movingparticles in and out of reagent streams (shown on the left of FIG. 3A),including a blow up of the texture of the obstacles on the chip (shownon the right of FIG. 3A). FIG. 3B illustrates the bump array 300 shownin FIG. 3A. 3 μm fluorescent beads 302 enter array at lower left and arebumped through a simulated reagent stream marked with non-bumpable 0.5μm beads 304.

FIG. 4A-H is a series of photographs depicting the lysis of an E. colicell in a bump array. Spheroplasted cell expressing fluorescent protein(“GFP”) was stained with fluorescent DNA dye and bumped into SDS lysisstream located in top half of flow cell. Comet tail is released GFP.Nucleoid remains compacted and continues to bump diagonally. GFP isbelow critical size, and travels with flow.

FIG. 5 illustrates an exemplary system 500 for processing of a fluidsample, according to some implementations of the current subject matter.The system 500 can process a blood sample 502 as input. The system 500can include at least one input reservoir 508, a bump array 512, and atleast one output reservoir 518. The components of system 500 can beimplemented in a single housing, separate housings that can be connectedto one another, and/or in any other fashion. The bump arrays 512 can bea bump array shown in FIG. 1 and/or a plurality of bump arrays shown inFIG. 1 that can be sequentially coupled and/or connected to one anotherfor the purposes of processing blood sample 502. The fluid sample canflow in a direction 520 from the input reservoirs 508 to outputreservoirs 518, as shown in FIG. 5. The input reservoirs 508 can includereservoirs of phosphate buffered saline (“PBS”) 510, on either side ofthe blood input port, a cell lysis buffer 504, and a wash buffer 506.The blood sample enters at the separate input port (or inlet) 540 and istreated to generate cells 516 (e.g., RBC, WBC, etc.) that enter the bumparray 512 for processing. In some implementations, the bump array caninclude a particular and separate input port 540 that is designed toreceive the blood sample 502. As such, the input port 540 (and otherinput ports shown and discussed in connection with FIGS. 6 and 7 below)can serve as a discrete input port that is physically separate from theother input reservoirs 504 and 506. The blood sample input port 502 canbe always physically distinct and discrete from the surrounding bufferinput ports so that the sample path is well defined and, further, sothat the desired product can come out in a relatively well definedposition at the bottom output region. In some implementations, theoutput reservoirs can be not so well-defined as the input reservoirs,except for a physically discrete output reservoir that receives thedesired output (as there is a desire to keep it from mixing with otherwaste streams). The cells are further lysed using the cell lysis buffer504 to generate nuclei 514 that are further processed in the bump array512. While the blood sample is being processed, the cells 516, thenuclei 514, and the remainder of the blood sample are being processed inthe bump array 512. The remainder of the blood sample gets deposited asplasma 522 in the output reservoir. The remainder of the lysing processin the cell lysis buffer 504 can be deposited in the lysis debris outputreservoir 526. The nuclei 514 are deposited in the nuclei outputreservoir 528 and the remainder of the wash buffer solution can bedeposited in the buffer output reservoir 524. System 500 can be used toperform processing operations 1002 and 1004 shown and discussed below inconnection with FIG. 10 and can be designed to purify cells (e.g.,leukocytes) from biological fluids, such as blood, and nuclei from lysedcells.

FIG. 6 illustrates another exemplary system 600 for processing a fluidsample, according to some implementations of the current subject matter.The system 600 can include two separate components or “chips” 602 and604 that can process fluid samples. The chip 602 can have a relativelylarge critical size for bumping that can be appropriate for the nucleisample input. The system 600 can be used to purify high molecular weight(“HMW”) DNA from isolated cell nuclei. The chip 602 can include inputreservoirs 612, a bump array 614, and output reservoirs 616. The chip602 can include a particle trap 611. The chip 602 can include a separateinput port (or inlet) 640 for receiving the nuclei. The chip 604 caninclude input reservoirs 632, a bump array 634, and output reservoirs636. The chip 604 can include a separate input port (or inlet) 641 forreceiving the materials that have been processed by the chip 602, i.e.,DNA+protein 621. The bump arrays 614 and 634 can be similar to the bumparray shown in FIG. 1. The general direction of the fluid flow throughthe chips 602, 604 is indicated by an arrow 620.

The chip 602 can include a nuclear wash buffer 610, a guanidineisothiocyanate (GuSCN) buffer 613, and a buffer 618 (which can besimilar to a wash buffer 506 shown in FIG. 5). The nuclei 615 enter theseparate input port 640 and are then processed through the GuSCN buffer613 in the bump array 614. The GuSCN can dissociate the nuclei andremove all nuclear proteins 619 from the DNA. The DNA and proteins havea smaller effective diameter than the critical size for bump array 602,and they flow out of the array with the GuSCN lysis reagent stream atoutlet reservoir 621, as any unlysed nuclear material 623 is directed tothe particle trap 611. HMW DNA and protein 619 flow into DNA+proteinoutput reservoir 621, which is then routed for further processing to thesample input reservoir channel of chip 604.

The HMW DNA and protein 619 can be received as nuclear lysate from thechip 602 in the input reservoirs 632 (and, in particular, the inputreservoir 641). Similar to FIG. 5, the input reservoirs 640 and 641 inthe chips 602 and chip 604, are specifically designated to preventmixing with any other materials present. The bump array of 604 can havea smaller critical size appropriate for bumping HMW DNA in the lysedsample. The DNA can be bumped rightward into DNA buffer (typically about10-50 mM Tris-HCl, pH 7.5-8.0, and about 1-5 mM EDTA) away from theGuSCN and denatured nuclear proteins which flow with the bulk fluid path(down). The DNA 635 can exit the bump array at output reservoir 637 ofthe chip 604's output reservoirs 636. The remainder of the waste lysisstream and wash buffer can be deposited in the buffer output reservoir639. The system 600 can be used to perform operation 1006 shown anddiscussed below in connection with FIG. 10.

FIG. 7 illustrates another exemplary system 700 for processing of afluid sample, according to some implementations of the current subjectmatter. The system 700 can include input reservoirs 710, a separateinput port (or inlet) 740 for receiving HMW DNA sample, a bump array720, and output reservoirs 730. Each input reservoir 710 can be discreteto prevent dilution and/or mixing of components. The general directionof the fluid sample flow in the system 700 is indicated by the arrow740. The input to the system 700 can be HMW DNA 702, which can bereceived in the separate input port 740 and is bumped through bufferentering the array immediately to the right of the input port. The HMWDNA 702 can then enter transposase reagent stream 704 where the HMW DNAcan be modified with sequencing library adapters as shown in FIG. 8. Asshown in FIG. 8, reaction with the transposon reagent of the currentsubject matter, produces HMW co-integrates that are still bumpable. Theco-integrates 712 are bumped into a wash buffer region fed by reservoir715, in order to remove free adapter substrates and transposase from theDNA. Then, the treated material can be further bumped into a restrictionenzyme reagent stream 706 to which cleaves the adapters at engineeredpositions (see FIG. 8B) to liberate the final sequencing library. Thefinal library is low in molecular weight and therefore no longerbumpable. As a result, the final library 716 can exit the bump array inthe restriction enzyme reagent stream 716. Treatment with reagent 706can result in the final low molecular weight library and reagent 716deposited in an output port (or outlet) that can be specificallydesignated to receive the final LMW library+reagent and to preventmixing of the final LMW library+reagent with any other output compoundsof this process. HMW DNA that is not processed by thetransposase/restriction enzyme streams remains HMW and can pass into theparticle trap 714 at the far right side of the array. System 700 can beused to perform operation 1008, 1010, 1012, 1014 shown and discussed inconnection with FIG. 10.

FIG. 8A is a schematic diagram illustrating a transposition-mediatedlibrary generation system 800 that can be optimized for use in automatedbump array instrument. Transposase enzymes can be loaded with linearrecombinant transposon substrates. Lines 802 and 804 represent dsDNAsubstrates. Transposition reactions can insert the linear transposonsubstrates into the HMW DNA targets. The co-integrate product DNA canremain high in molecular weight after transposition, and therefore itcan be bumped away from the transposon reagent stream after reaction.FIG. 8B is a schematic diagram depicting the structure of transpositioncointegrates. NGS adapter sequences can be located adjacent to thetransposon ends and allow sequencing into the insert from either end.The final library can be cleaved out of the HMW co-integrate by cleavageat engineered restriction enzyme sites that release theadapter-terminated sequencing library from the rest of the co-integrate(final cleavage not shown).

Although production of NGS libraries can be one of the exemplaryembodiments of the current subject matter, there can be many otherapplications of the system. FIG. 9 illustrates this point schematically,shown an exemplary system 900 that can utilize multiple bump array stepin serial fashion for performing multiple sequentially orderedprocessing steps on a fluid sample, according to some implementations ofthe current subject matter. The system 900 can have components that canbe similar to the components in any of the systems shown and describedin connection with FIGS. 1-7 above. The system 900 can include an inputsorting array 902, a first treatment mechanism 904, a second treatmentmechanism 906, and a size fractionation array 908. The fluid sample thatenters the system 900 can flow in a general direction 920. The sampleinput that enters the input sorting array 902 can be washed and/orsorted using at least one reagent, where various “cells of interest” canbe produced. Other components can be deposited in a buffer. The cells ofinterest can be treated using the first treatment mechanism 904, whichcan include a first reagent and an appropriate buffer. Treatment by thefirst treatment mechanism 904 can result in a first treatedsample-reagent combination. The first treated sample-reagent combinationis then processed by the second treatment mechanism 906, which caninclude a second reagent and an appropriate buffer. Treatment by thesecond treatment mechanism 906 can result in a second treatmentsample-reagent combination that is passed on to a size fractionationarray 908, which can sort the combination from small to large particlesaccordingly.

The system can retain flexibility regarding how these processes can becombined. Each of the five processes can be carried out insingle-function flow chips that can be chained together serially. Inthis configuration, the product of each chip can be fed into the inputport of the subsequent chip. Alternatively, multiple steps can becarried out on the same chip by using multiple reagent streams separatedlaterally across the chip, and changing the array dimensions laterallyacross the chip to match the size of the intermediates that need to bebumped at each stage.

FIG. 10 illustrates a method 1000 for sequentially processing bloodsamples using bump arrays, according to some implementations of thecurrent subject matter. At 1002, cells can be separated from abiological fluid, such as whole blood. A few microliters (“μl”) of wholeblood can be obtained. Blood cells can be separated from plasma. Cellscan be washed with a buffer stream as they are separated from theplasma. At 1004, cells can be lysed. Washed cells can be lysed bybumping them through a reagent stream containing non-ionic detergent.After removing the lysis reagent, intact leukocyte nuclei can be bumpeddiagonally through a wash buffer stream. Cytoplasmic contents are toosmall to bump and can be carried out of the array in the detergent lysisstream. At 1006, chromosomal DNA can be isolated and/or purified. Washedleukocyte nuclei can be bumped through a nuclear lysis reagent stream toremove all lipid and nuclear proteins from the HMW chromosomal DNA. Thearray dimensions can be chosen so that HMW DNA, in its double-strandedrandom-coil configuration, can be bumped diagonally out of the lysisreagent stream. All nuclear lipids, RNA, and proteins may be too smallto bump, and can be carried out of the array in the lysis stream. At1008, purified DNA can be reacted with a transposase-adapted reagent togenerate library cointegrates. Purified HMW DNA can be bumped through areagent stream containing a transposase complex that can be preloadedwith sequencing-adapter-modified transposon ends. The current subjectmatter can provide a transposase complex in which the transposon-adaptedends of the transposasome can be on the same linear piece of DNA. As aresult, in this system, a reaction of the transposasome with the HMW DNAcan generate a colinear insertion product that can increase the size ofthe HMW DNA target. The target DNA can remain bumpable, and, thus, thetarget DNA can be separated from the transposasome reagent stream andunreacted adapter DNA. At 1010, HMW cointegrates can be purified fromthe transposase reagent stream. At 1012, cointegrates can be reactedwith restriction enzyme to generate a sequencing library. At 1014, thelibrary can be separated from uncut HMW DNA and recovered from the bumparray. In some implementations, the final sequencing library can becleaved from the HMW co-integrate DNA by bumping the DNA through arestriction enzyme reagent stream. The enzyme can cleave engineeredsites in the modified transposon that lie just outside of the sequencingadapters. The cleaved library can be low in molecular weight ˜-200-2000bp), and can be no longer bumpable. The library can be removed from thearray in the restriction enzyme stream. Uncleaved, unreacted HMW DNA canbe bumped out of the reagent stream diagonally (and can be recovered).

In some implementations, the current subject matter system can includemicron-sized post arrays with high structural rigidity and high aspectratios for the purposes of processing fluid samples. The bump arrays canbe manufactured from silicon, cyclic olefin resin, molded plasticdisposable flow cells, as well as any other materials.

In some implementations, bump arrays and systems can separate orfractionate, analyze, and/or collect purified or processed polynucleicacid analytes or fractions derived from a raw biological sample.

In some implementations, the current subject matter can also processsmaller nucleic acid molecules by attaching the nucleic acids tomicroparticles that can be bumped in a bump array. The microparticlescan act as carriers for transporting the attached nucleic acids throughreagent streams for modification of the nucleic acids. For example,emulsion PCR with primer-modified microparticles can be used forgeneration of DNA sequencing template beads (Ion Torrent and 454sequencing methods; Rothberg et al. 2011. Nature v475, pp 348-352;Margulies et al., Nature. V437, pp 376-380). In some implementations,emulsion PCR methods can be used for evaluation of the frequency of raremutant genes in tissue from cancer patients (Vogelstein's “BEAMing”method, Diehl et al. Nature Methods. 2006 v3 pp 551-559). In someimplementations, the current subject matter can be used to processparticle-based emulsion PCR by combining washing, denaturation, andprimer hybridization into a single bump array process. For example, abump array can be designed with post spacing chosen so that the criticaldiameter of the array can be less than that of the microparticles usedfor the emulsion PCR. This can ensure that the microparticles can bebumped consistently at all positions within the array. After emulsionPCR, the emulsion is broken and the aqueous particles fraction is fedinto the array near the upper left hand corner. As the particles enterthe array, they can be bumped rightward, while the PCR reagents can flowdownward in the direction of bulk flow (the directional flow can besimilar to the one shown in FIGS. 5-7, for example). A suitable washbuffer can be fed into the top of the array immediately to the right ofthe particle input port. As the particles are bumped out of the inputstream, they can pass through the wash buffer stream, which can cleanaway additional PCR reagent. As the particles move further down thearray, they can enter a denaturing reagent stream (e.g., which cancontain about 20-200 mM KOH or NaOH with about 1-10 mM EDTA), which canconvert the double-stranded amplicons on the particles tosingle-stranded form. The non-covalently bound amplicon strand can bewashed down the array with the denaturant stream, and the particles canbe bumped rightward into a neutralizing buffer that can be suitable forhybridization reactions in the next step. Generally, such neutralizingbuffer can contain a buffer (for example, 20-200 mM Tris-HCl, pH7.5-8.0) and monovalent ions to support hybridization (for example,20-500 mM NaCl). The particles can then be bumped through ahybridization reagent stream containing sequencing primer (in the caseof the 454/Ion Torrent applications), or labeled oligonucleotide probe(in the case of the BEAMing application). The reagent stream can haveoligo probes in the low micromolar concentration range (about 0.1micromolar to about 50 micromolar), and can have about the same ionicstrength as the neutralizing buffer stream described above. The ionicstrength can be adjusted higher or lower as needed to achieve thecorrect stringency of hybridization. In the final processing step of thearray, the hybridized particles are bumped out of the hybridizationstream into a final wash buffer stream. This final wash buffer is chosenaccording to the downstream application to be used (sequencing in thecase of 454/Ion applications, fluorescent particle sorting in the caseof the BEAMing assay). The hybridized, washed particles are collectedfrom the output port of the final wash buffer located near the lowerright corner of the array.

EXAMPLES Example 1 Bump Array Process for Purification of LeukocyteNuclei from Whole Blood

A single array performs operations 1002 and 1004, shown in FIG. 10,encompassing separation of cells from plasma and isolation of leukocytenuclei, respectively (as shown in FIG. 5). The array, shownschematically in FIG. 5, is designed to bump all particles >3-4 μm indiameter. Setting the critical dimension at this size bumps cells (redblood cells (RBCs) 6-8 μm, white blood cells (WBCs), 6-16 μm) and nuclei(5-9 μm) diagonally across the flow direction. Blood flows into a regionof the array filled with buffered saline. As the cells bump through thisregion, they are washed free of plasma. Continuing laterally across thearray, the cells are bumped into a stream of lysis buffer containing aconcentration of non-ionic detergent sufficient to lyse RBCs and WBCs,but not WBC nuclei. Since the nuclei are still larger than the criticaldiameter of the array (3-4 mm), they follow the same diagonal path asthe cells. The nuclei exiting the lysis buffer pass into a stream ofwash buffer (lysis buffer without detergent, and are collected in anoutput reservoir near the bottom right corner of the array.

The post spacing of this array is based on a simplified version of thecell sorting array (“FD” device) of Davis et al., 2006. Proc Natl AcadSci USA. v103, pp 14779-84, incorporated herein by reference in itsentirety. That device utilized a constant post diameter (22 μm) andconstant gap size (“G” in FIG. 1, 10 μm), but varied the row offsetdistance (“d” in FIG. 1) in stepwise fashion from small to large values.The smallest offset distance used in the Davis array, approximately 0.5μm, gives a critical particle diameter for bumping of ˜3 μm. This designis appropriate for this nuclear isolation array of this system, becausethe smallest diameter expected for nuclei (and cells) is ˜5 μm. Forthese reasons, this system may use a constant offset of 0.5 μmthroughout the array.

The overall dimensions of the array are also derived from the Davissorting array, since the throughput of that array is a good match forDNA sequencing applications. The processing speed of that device was 1μl of whole blood per hour. This corresponds to about 66 ng of genomicDNA per hour (10⁴ WBC/μl×0.0066 ng DNA/cell). Standard DNA inputrecommendations for NGS library protocols have been decreasing steadily,and some practitioners can reproducibly produce libraries with 50-100 nggenomic DNA.

The concentration and type of detergent can be adjusted as needed tooptimize nuclear yield. The relative array areas devoted to the variouschip inputs will be investigated to optimize nuclear yield and purity.For instance, the width of the lysis buffer stream can be adjusted toalter the residency time of cells or nuclei in the lysis reagent.Similarly, the width of the wash buffer stream can be widened to providea more stringent removal of detergent or lysed blood components from thenuclei. To facilitate investigation of these issues, early chipprototypes will be equipped with many regularly spaced input reservoirs.In such prototypes, the width of any reagent stream can be widened byfilling additional adjacent input reservoirs with the reagent.

Example 2 Bump Array Process for Purification of HMW DNA from IsolatedLeukocyte Nuclei

The next process is purification of DNA from the cell nuclei. The systemaccomplishes this task using chaotropic salt solutions (4 M guanidinethiocyanate, GuSCN) to lyse nuclei and dissociate chromosomal proteinsfrom the DNA. Lysis with chaotropes is faster and more complete thanpopular SDS-proteinase K protocols. Silicon arrays are chemically coatedwith fluorosilane to prevent DNA binding to the silicon oxide surfacesin the presence of high concentrations of chaotrope.

A pair of bump arrays with different post geometries is used for thisprocess (as shown in FIG. 6). The first array has a geometry appropriatefor bumping nuclei (critical particle diameter=˜3-4 μm). In this region,the input stream of nuclei is passed into the GuSCN stream where thenuclei are lysed. DNA and dissociated nuclear proteins are smaller thanthe critical particle diameter, and flow with the lysis reagent. Largeparticles (>3-4 μm) such as partially unlysed nuclei, are bumpedrightward and are trapped in a particle trap at the right edge of thedevice. This trap is a serpentine fluidic channel with many entry pointsrunning the length of the array. Unwanted particles enter the trapchannel and remain there slowly traversing the long channel for theduration of the DNA purification process. The lysis stream carrying theDNA and denatured protein is piped into the input port of the secondarray. The second array has a critical particle diameter of around 0.6μm, which bumps double-stranded linear molecules >˜40 kb. As a result,HMW DNA is bumped out of the GuSCN stream, and is washed in a bufferstream as it travels rightward to the collection reservoir at the lowerright corner.

A key issue for this process is how the nuclei behave as they begin tolyse. There is a risk that extremely large, chromosome-sized DNAmolecules (>200 kb), that may spill out of partially lysed nuclei, couldbecome entangled with the array posts and/or other nuclei, and clog thearray. A related issue is whether such extremely large DNA molecules canclog the arrays, even in purified form. To overcome these issues, theaverage size of the nuclear DNA can be reduced before lysis, by, forexample, treating the nuclei with a low concentration of either anuclease or a chemical cleavage agent. Preferably, the average DNA sizeis reduced to between 50 and 200 kb, where bump array technology workswell. Preferred reagents would be double-strand endonucleases likemicrococcal nuclease or rare-cutting restriction enzymes. Optionally,the cleavage reagent stream is positioned immediately to the right ofthe nuclear sample input in the first array (left of the GuSCN inputstream), so that the cleavage agent is washed in and out of the nucleibefore entering the lysis stream. Alternatively, a cleavage process stepis inserted into the array of Example 1 (which illustrates operation1002 shown in FIG. 10).

Another important parameter in this process is the time of exposure tothe GuSCN reagent needed to obtain efficient lysis. The width of theGuSCN layer, the bump angle of the array (adjustable by changing gap andoffset of the array), and the flow rate are critical variables that aremanipulated to vary exposure time. The lysis process is monitored inreal time using microscopy of nuclei passing through the arrays. DNArecovery and purity are measured using standard DNA and protein assays.

Example 3 Bump Array Process for NGS Library Formation

Purified HMW genomic DNA from the arrays of Example 2 (which illustratesoperations 1006 and 1008 shown in FIG. 10) is passed to an array thatperforms a transposition-mediated library formation reaction (as shownin FIG. 7). The array geometry of the library array is similar to thatof the second array of Example 2 (which illustrates operation 1008 shownin FIG. 10): DNA bigger than ˜40 kb is bumped rightward into atranposase-based library formation reagent stream (as shown in FIG. 7).

This library formation reaction is a modification of the Nextera libraryconcept (Epicentre Biotechnologies/Illumina). In contrast to the Nexterasystem, this system uses recombinant transposon substrates carrying bothtransposon ends on the same linear double-stranded DNA (dsDNA) molecule(as shown in FIGS. 8A-B). The transposition reaction inserts the entirerecombinant transposon into the HMW genomic target DNA. The criticalfeature of this reaction is that the co-integrate product remains highin molecular weight, and, therefore, the bump array process can be usedto purify the reaction products away from free transposase and unreactedtransposon substrates.

After bumping out (i.e., removing) of the transposase reagent stream,the co-integrate DNA is washed in a buffer stream, and bumped into arestriction enzyme reagent stream. The restriction enzyme cleaves justoutside of the NGS adapter sequences on the 5′ sides of the transposonends (as shown FIGS. 8A-B). The final library is low in molecular weight(fragments ranging ˜200-2000 bp), and is recovered from the array in therestriction enzyme stream. Additional purification of the library can beperformed to remove the restriction enzyme prior to loading the libraryon the sequencer.

Transposase enzymes suitable for NGS library construction arecommercially available (Nextera, a mutant Tn5 transposase). Epicentrealso sells linear Tn5 transposon substrates for insertional mutagenesisand Sanger sequencing (see EZ-Tn5™<oriV/KAN-2> Transposon InsertionKit). These linear substrates are used for initial testing of theproposed bump array process. For instance, the Tn5-KAN-2 commercialsubstrate can be inserted into a defined HMW target such as phage lambdaDNA. Transposition efficiency is assessed by electrophoresis,restriction mapping (the transposon and lambda each have single sitesfor the enzyme Xho I), or blot hybridization of the product DNA.

For commercialization, recombinant transposon substrates with Tn5 ends,NGS adapter sequences, and rare-cutting restriction sites for libraryrelease are constructed using standard recombinant DNA methods.Alternatively, other high activity in vitro transposition systems, suchas Tn552 from S. aureus may be used.

Operation 1014 shown in FIG. 10 can optimize the two enzymatic libraryconstruction reactions for efficient utilization of genomic DNA (andreagents). As mentioned previously, reagent concentration, bump angle,reagent stream width, post array spacing, and flow rate can all beadjusted to optimize the reagent-DNA contact time.

Example 4 Bump Array Process for Isolation of Bacterial DNA from a HumanBlood Sample

Step 1.

A sample of human blood is treated with non-ionic detergent underconditions where RBCs and WBCs are lysed, but WBC nuclei remain intact(0.32 M sucrose, 5 mM MgCl2, 1% Triton X-100, 0.01 M Tris-HCl, pH 7.6).These conditions are not strong enough to lyse bacteria, and they remainin the lysate as intact cellular forms.

Step 2.

The lysate is fed into a first post array designed to bump particles 3-4microns in diameter. The post array of Example 1 has suitable spacingfor this application. The lysate is fed into the array near the left topcorner. Large particles including WBC nuclei and partially lysed humancells are bumped rightward, while smaller particles, including bacterialcells, travel in the same direction at the bulk fluid flow, straightdown the left side of the array, and are recovered from the bottom leftside of the array.

Step 3.

The small particle output of the first array (from left side), is fedinto a second post array that is designed to bump bacterial cells(critical diameter for bumping approximately 0.7 microns). Design ofsuch arrays is described in Morton et al., 2008 (Morton K J, LoutherbackK, Inglis D W, Tsui O K, Sturm J C, Chou S Y, Austin R H. 2008. LabChip. v8, pp 1448-1453, incorporate herein by reference in itsentirety). The small particle lysate from the first array is fed intothe array near top left corner. The second array is designed to havethree separate reagent streams entering the array on the right side ofthe lysate input port. The four input ports for sample and reagents(reagent streams 1-3) are separated by wash buffer ports, so that lowmolecular weight compounds are washed from the bumped components beforeentering the next reagent stream.

Step 4.

As the lysate flows into the second post array, bacterial cells arebumped rightward from the lysate stream into isotonic wash buffer (50 mMTris-HCl, pH 8.0, 150 mM NaCl, 1.125 M sucrose; Morton et al., 2008). Asthe bacterial cells flow further down the array, they are bumpedrightward into the first reagent stream, which contains enzymes thatdegrade bacterial cell walls (hen egg white lysozyme, mutanolysin) anddetergents to lyse the bacterial membrane in isotonic wash buffer (0.4mg/ml lysozyme and mutanolysin, 8% weight/volume sucrose, 10 mM EDTA, 1M NaCl, 0.5% Brij 58, 0.2% deoxycholate). Bacterial cells will be lysedin this reagent stream, but the bacterial chromosome will remain in acompacted, bumpable form, known as a nucleoid (Worcel A, Burgi E. 1972.J. Mol. Biol. v71, pp 127-147, incorporated herein by reference in itsentirety). As the nucleoids flow further into the array, they are bumpedinto a buffer stream which removes components of the first reagentstream and conditions the nucleoids for the next reagent stream (50 mMTris HCl, 150 mM NaCl, pH 7.9).

Step 5.

The nucleoids are bumped into a second reagent stream which contains arestriction enzyme with a rare sequence specificity (such as the enzymesNot I or Sfi I, both available from New England Biolabs), in a bufferthat will support enzyme activity (for Not I, conditions are 50 mM TrisHCl, 150 mM NaCl, 10 mM MgCl2, pH 7.9, 100 micrograms/ml bovine serumalbumin, 1 mM dithiothreitol). This treatment cleaves the bacterialchromosome into fragments ranging in size between 40 kb and 1000 kb(Smith C L, Econome J G, Schutt A, Klco S, Cantor C R. 1987. Science.v236, pp 1448-1453, incorporated herein by reference in its entirety),but will leave the nucleoids in compacted form and associated withpackaging proteins. The purpose of the restriction cleavage is to reducethe average DNA fragment size in the nucleoids so that the chromosomalDNA will not clog the array in the next step, in which the packagingproteins are stripped from the DNA. As the still-folded nucleoids flowdown the array they are bumped out of the second reagent stream into awash buffer without enzymes (50 mM Tris HCl, pH 7.9, 150 mM NaCl).

Step 6.

The nucleoids are bumped into a third reagent stream containing a highconcentration of chaotrope (4 M guanidine isothiocyanate). Thistreatment completely dissociates the nucleoids into free protein andDNA. The majority of the bacterial DNA will be in fragments that aregreater than 40 kb. Linear DNA molecules of this size will behave asparticles with a diameter of around 1 micron (Robertson R M, Laib S,Smith D E. 2006. Proceedings of the National Acad USA. v103, pp7310-7314, incorporated herein by reference in its entirety), andtherefore they will bump in the array to the right, just as thebacterial cells and nucleoid did. The associated nucleoid proteins aretoo small to be bumped by the array and will travel straight down thearray with the chaotrope reagent stream. The DNA is bumped rightward outof the chaotrope reagent stream and into a final buffer suitable for DNAstorage, or alternatively, a buffer suitable for the next processingsteps, as appropriate. The purified final DNA products are collectedfrom channels exiting the array near the lower right corner.

Step 7.

Optionally, the purified DNA from Step 6 can be fed into an array asdescribed in Example 1 for generation of DNA sequencing libraries. Thesequence information obtained can be used to diagnose infections, andalso guide treatment decisions by revealing drug resistances andsensitivities of the infecting organism.

In some implementations, the current subject matter can be implementedtogether with the use of various computing systems and/or computerprogram products. Such systems and products can be used to process,monitor, collect, and/or otherwise assist the various components of thecurrent subject matter's system. Such computer program products cancomprise non-transitory computer readable media storing instructions,which when executed one or more data processor of one or more computingsystems, causes at least one data processor to perform operationsherein. Similarly, such computer systems can include one or more dataprocessors and a memory coupled to the one or more data processors. Thememory may temporarily or permanently store instructions that cause atleast one processor to perform one or more of the operations describedherein. In addition, methods can be implemented by one or more dataprocessors either within a single computing system or distributed amongtwo or more computing systems.

The computing systems and/or products that can be used in conjunctionwith the systems and methods disclosed herein can be embodied in variousforms including, for example, a data processor, such as a computer thatalso includes a database, digital electronic circuitry, firmware,software, or in combinations of them. Moreover, the above-noted featuresand other aspects and principles of the present disclosedimplementations can be implemented in various environments. Suchenvironments and related applications can be specially constructed forperforming the various processes and operations according to thedisclosed implementations or they can include a general-purpose computeror computing platform selectively activated or reconfigured by code toprovide the necessary functionality. The processes disclosed herein arenot inherently related to any particular computer, network,architecture, environment, or other apparatus, and can be implemented bya suitable combination of hardware, software, and/or firmware. Forexample, various general-purpose machines can be used with programswritten in accordance with teachings of the disclosed implementations,or it can be more convenient to construct a specialized apparatus orsystem to perform the required methods and techniques.

The systems and methods disclosed herein can be implemented as acomputer program product, i.e., a computer program tangibly embodied inan information carrier, e.g., in a machine readable storage device or ina propagated signal, for execution by, or to control the operation of,data processing apparatus, e.g., a programmable processor, a computer,or multiple computers. A computer program can be written in any form ofprogramming language, including compiled or interpreted languages, andit can be deployed in any form, including as a stand-alone program or asa module, component, subroutine, or other unit suitable for use in acomputing environment. A computer program can be deployed to be executedon one computer or on multiple computers at one site or distributedacross multiple sites and interconnected by a communication network.

As used herein, the term “user” can refer to any entity including aperson or a computer.

Although ordinal numbers such as first, second, and the like can, insome situations, relate to an order; as used in this document ordinalnumbers do not necessarily imply an order. For example, ordinal numberscan be merely used to distinguish one item from another. For example, todistinguish a first event from a second event, but need not imply anychronological ordering or a fixed reference system (such that a firstevent in one paragraph of the description can be different from a firstevent in another paragraph of the description).

The foregoing description is intended to illustrate but not to limit thescope of the embodiments of the disclosure, which is defined by thescope of the appended claims. Other implementations are within the scopeof the following claims.

To provide for interaction with a user, the subject matter describedherein can be implemented on a computer having a display device, such asfor example a cathode ray tube (CRT) or a liquid crystal display (LCD)monitor for displaying information to the user and a keyboard and apointing device, such as for example a mouse or a trackball, by whichthe user can provide input to the computer. Other kinds of devices canbe used to provide for interaction with a user as well. For example,feedback provided to the user can be any form of sensory feedback, suchas for example visual feedback, auditory feedback, or tactile feedback;and input from the user can be received in any form, including, but notlimited to, acoustic, speech, or tactile input.

The implementations set forth in the foregoing description do notrepresent all implementations consistent with the subject matterdescribed herein. Instead, they are merely some examples consistent withaspects related to the described subject matter. Although a fewvariations have been described in detail above, other modifications oradditions are possible. In particular, further features and/orvariations can be provided in addition to those set forth herein. Forexample, the implementations described above can be directed to variouscombinations and sub-combinations of the disclosed features and/orcombinations and sub-combinations of several further features disclosedabove. In addition, the flows depicted in the accompanying figuresand/or described herein do not necessarily require the particular ordershown, or sequential order, to achieve desirable results. Otherimplementations can be within the scope of the following claims.

Example embodiments of the methods and components of the presentdisclosure have been described herein. As noted elsewhere, these exampleembodiments have been described for illustrative purposes only, and arenot limiting. Other embodiments are possible and are covered by thepresent disclosure. Such embodiments will be apparent to persons skilledin the relevant art(s) based on the teachings contained herein. Thus,the breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

1. A method for processing of a biological fluid, comprising: separatingat least one first cell from the biological fluid; applying at least onefirst treatment to the at least one separated cell to produce a firsttreated solution; applying at least one second treatment to the firsttreated solution to produce a second treated solution; and processing atleast one of the first treated solution and the second treated solutionusing a deterministic lateral displacement to generate an outputsolution.
 2. The method according to claim 1, wherein the biologicalfluid includes at least one of the following: whole blood, urine, spinalfluid, saliva, buccal swabs, sputum, bronchial lavage, gastric lavagefluid, microbial culture media, feces, buffy coat, serum, plasma,platelet concentrate, water samples, and/or any other biological,chemical, and/or biochemical fluids and/or any combination thereof. 3.The method according to claim 1, wherein the deterministic lateraldisplacement uses at least one bump array to process at least one of thefirst treated solution and the second treated solution.
 4. The methodaccording to claim 1, wherein the deterministic lateral displacementuses a sequential arrangement of a plurality of bump arrays to processat least one of the first treated solution and the second treatedsolution.
 5. The method according to claim 1, wherein the biologicalfluid is whole blood; the applying at least one first treatment includeslysing cells separated from the whole blood to generate a purifieddeoxyribonucleic acid (“DNA”); and the applying at least one secondtreatment includes combining the purified DNA with a transposase complexand at least one sequencing adaptor.
 6. The method according to claim 1,further comprising fractionating the output solutions based on a size ofat least one cell contained within the output solution.
 7. A system forprocessing of a biological fluid, comprising: at least one inputreservoir for receiving the biological fluid and separating at least onefirst cell from the biological fluid; at least bump array mechanismcoupled to the at least one input reservoir for applying at least onefirst treatment to the at least one separated cell to produce a firsttreated solution; applying at least one second treatment to the firsttreated solution to produce a second treated solution; and processing atleast one of the first treated solution and the second treated solutionusing a deterministic lateral displacement to generate an outputsolution; and an output reservoir for receiving the output solution. 8.The system according to claim 7, wherein the biological fluid includesat least one of the following: whole blood, urine, spinal fluid, saliva,buccal swabs, sputum, bronchial lavage, gastric lavage fluid, microbialculture media, feces, buffy coat, serum, plasma, platelet concentrate,water samples, and/or any other biological, chemical, and/or biochemicalfluids and/or any combination thereof.
 9. The system according to claim7, wherein the at least one bump array uses a deterministic lateraldisplacement to process at least one of the first treated solution andthe second treated solution.
 10. The system according to claim 7,further comprising a sequential arrangement of a plurality of bumparrays to process at least one of the first treated solution and thesecond treated solution.
 11. The system according to claim 7, whereinthe biological fluid is whole blood; the applying at least one firsttreatment includes lysing cells separated from the whole blood togenerate a purified deoxyribonucleic acid (“DNA”); and the applying atleast one second treatment includes combining the purified DNA with atransposase complex and at least one sequencing adaptor.
 12. The systemaccording to claim 7, wherein the output reservoir fractionates theoutput solutions based on a size of at least one cell contained withinthe output solution.
 13. A method for processing a whole blood sampleusing a sequential and continuous arrangement of bump arrays integratedin a continuous flow operation, comprising: receiving the whole bloodsample at a first bump array in the arrangement of bump arrays;purifying the hole blood sample to produce white blood cells; isolatingnuclei from the white blood cells; isolating deoxyribonucleic acid(“DNA”) from the nuclei; purifying DNA from the nuclei; and treating thepurified DNA using at least one chemical and/or enzymatic DNA treatment.14. method for processing of a fluid sample using at least one bumparray, comprising receiving the fluid sample at the at least one bumparray; isolating, using the at least one bump array, at least onenucleic acid-containing cell and/or particle of interest from the fluidsample on the basis of a size of the cell and/or particle; contactingusing the at least one bump array, the isolated cell and/or particlewith at least one reagent stream for releasing at least one nucleic acidfrom the cell and/or particles in substantially pure form; moving, usingthe at least one bump array, the at least one purified nucleic acid outof the reagent stream; and removing the at least one purified nucleicacid from the at least one bump array.
 15. The method according claim14, wherein a plurality of bump arrays processes the nucleic acid,wherein individual bump arrays in the plurality of bump arrays areconnected in series, wherein an output of one bump array is provided toan input of a subsequent individual bump array.
 16. The method accordingto claim 14, wherein a single bump array is used for the receiving, theisolating, the contacting and the moving.
 17. The method according toclaim 14, wherein the fluid sample includes at least one of following:an avian whole blood and a mammalian whole blood; and wherein thenucleic acid-containing cells and/or particles are white blood cells.18. The method according to claim 14, wherein the fluid sample includesat least one of the following: an avian whole blood and a mammalianwhole blood; and wherein the nucleic acid-containing cells and/orparticles are circulating tumor cells.
 19. The method according to claim14, wherein the fluid sample includes at least one of the following: anavian whole blood and a mammalian whole blood; and wherein the nucleicacid-containing cells and/or particles include at least one of thefollowing: white blood cells, bacteria, viruses, fungi, and parasiticprotozoans.
 20. The method according to claim 19, wherein the biologicalfluid includes at least one of the following: whole blood, urine, spinalfluid, saliva, buccal swabs, sputum, bronchial lavage, gastric lavagefluid, microbial culture media, feces, buffy coat, serum, plasma,platelet concentrate, water samples, and/or any other biological,chemical, and/or biochemical fluids and/or any combination thereof. 21.A method for serially processing of a high molecular weight (“HMW”)nucleic acid using at least one chemical and/or enzymatic reagent streamusing at least one bump array, wherein HMW nucleic acid has an effectivehydrodynamic radius that is greater than a critical size of the at leastone bump array, comprising: receiving the HMW nucleic acid at the atleast one bump array; and contacting the HMW nucleic acid with the atleast one chemical and/or enzymatic reagent stream, wherein the at leastone chemical and/or enzymatic reagent stream flows in a direction of theflow of the HMW nucleic acid through the bump array; wherein the HMWnucleic acid reacts with the at least one chemical and/or enzymaticreagent stream.
 22. A method for serial processing of a nucleic acidusing at least one chemical and/or enzymatic reagent stream using atleast one bump array, comprising: receiving the nucleic acid; flowingthe nucleic acid into the at least one bump array, contacting, using theat least one bump array, the nucleic acid with at least one chemicaland/or enzymatic reagent stream; modifying, using at least one chemicaland/or enzymatic reagent stream, the nucleic acid; and removing, usingthe at least one bump array, the purified nucleic acid from the at leastone chemical and/or enzymatic reagent stream.
 23. The method accordingto claim 23, wherein a plurality of bump arrays serially process thenucleic acid, wherein individual bump arrays in the plurality of bumparrays are connected in series, wherein an output of one bump array inthe plurality of bump arrays is input to the subsequent individual bumparray in the plurality of bump arrays.
 24. The method according to claim23, wherein a single bump array performs the flowing, the contacting,the modifying, and the removing.
 25. The method according to claim 23,wherein the nucleic acid is high molecular weight (“HMW”) nucleic acid.26. The method according to claim 23, wherein the nucleic acid is adeoxyribonucleic acid (“DNA”), wherein the DNA is bound to at least onemicroparticle for carrying the DNA through the bump array.
 27. Themethod according to claim 26, wherein the is bound using at least one ofthe following: covalent binding and non-covalent binding.
 28. A methodfor processing of a fluid sample using at least one bump array,comprising receiving the fluid sample at the at least one bump array;isolating, using the at least one bump array, at least one nucleicacid-containing cell and/or particle of interest from the fluid sampleon the basis of a size of the cell and/or particle; contacting, usingthe at least one bump array, the isolated cell and/or particle with atleast one reagent stream for releasing at least one nucleic acid fromthe cell and/or particles in substantially pure form; modifying thenucleic acid; moving, using the at least one bump array, the at leastone purified nucleic acid out of the reagent stream; and removing the atleast one purified nucleic acid from the at least one bump array. 29.The method according to claim 28, wherein the nucleic acid is highmolecular weight (“HMW”) nucleic acid.
 30. The method according to claim28, wherein the nucleic acid is a deoxyribonucleic acid (“DNA”), whereinthe DNA is bound to at least one microparticle for carrying the DNAthrough the bump array.
 31. The method according to claim 28, whereinthe DNA is bound using at least one of the following: covalent bindingand non-covalent binding.
 32. The method according to claim 28, whereinthe fluid sample is serially processed using at least one chemicaland/or enzymatic reagent stream using the at least one bump array. 33.The method according to claim 32, wherein the nucleic acid is modifiedusing at least one chemical and/or enzymatic reagent stream.
 34. Themethod according to claim 33, wherein the modified nucleic acid includesat least one of the following: a deoxyribonucleic acid (“DNA”)sequencing library and a recombinant DNA library.
 35. A reagent systemfor generating DNA sequencing libraries, comprising: a transposasereagent complexed with a linear DNA reagent, the DNA reagent havingtransposase recognition sequences and sequencing adapter sequences ateach end of the DNA reagent, whereby on reaction with a DNA moleculetargeted for sequencing, the transposase inserts the adapter-bearinglinear DNA reagent into the sequencing target to form a cointegratestructure, wherein the sequencing target is cleaved at a singleposition, and the ends of the cleaved target are joined to the ends ofthe adapter-bearing linear DNA reagent.